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A leading objective in biology is to identify the complete set of activities that each gene performs in vivo In this study, we have asked whether a genetic approach can provide an efficient means of achieving this goal, through the identification and analysis of a comprehensive set of separation-of-function ( sof  - ) mutations in a gene. Toward this goal, we have subjected the Saccharomyces cerevisiae EST1 gene, which encodes a regulatory subunit of telomerase, to intensive mutagenesis (with an average coverage of one mutation for every 4.5 residues), using strategies that eliminated those mutations that disrupted protein folding/stability. The resulting set of sof  - mutations defined four biochemically distinct activities for the Est1 telomerase protein: two temporally separable steps in telomerase holoenzyme assembly, a telomerase recruitment activity, and a fourth newly discovered regulatory function. Although biochemically distinct, impairment of each of these four different activities nevertheless conferred a common phenotype (critically short telomeres) comparable to that of an est1 -∆ null strain. This highlights the limitations of gene deletions, even for nonessential genes; we suggest that employing a representative set of sof  - mutations for each gene in future high- and low-throughput investigations will provide deeper insights into how proteins interact inside the cell.

Using Separation-of-Function Mutagenesis To Define the Full Spectrum of Activities Performed by the Est1 Telomerase Subunitin Vivo